Amino Acids

Chapter 4
Amino Acids
. . . . . . . . . . . . . . . . . . . . . . . .
Chapter Outline
v Amino acids: Tetrahedral alpha (α) carbon with H, amino group, carboxyl group, and side chain
v Amino acids in proteins joined by peptide bonds
v Classification
Nonpolar: Ala, val, leu, ile, pro, met, phe, trp
Polar uncharged: Gly, ser, thr, tyr, asn, gln , cys
Acidic: Asp, glu
Basic: His, lys, arg
v Modified amino acids: Hydroxylysine, hydroxproline, thyroxin, methyl histidine, methyl
arginine, methyl lysine, γ−carboxyglutamic acid, pyroglutamic acid, aminoadipic acid
v Small molecules derived from amino acids: GABA, histamine, serotonin, β−alanine,
epinephrine, penicillamine, ornithine, citrulline, betaine, homocysteine, homoserine
v Ionic properties of amino acids
α−Carboxyl group: pKa’s range from 2.0 to 2.4
α−Amino group: pKa’s range from 9.0 to 9.8
Side chains
› Acidic pKa’s for asp, glu
› Basic pKa’s for his, arg, lys
v Amino acid chemistry
Carboxyl chemistry
Amino chemistry
Side chain chemistry
v Chirality: asymmetric carbon has four different groups attached
Optically active compounds
› Dextrorotatory (+), clockwise rotation
› Levorotatory (-), counterclockwise rotation
Asymmetric carbons
› One asymmetric carbon: Mirror image isomers or enantiomers
› More than one asymmetric carbon: enantiomers and diastereomers
D,L System: Typically naturally occurring amino acids: L-amino acids; related to Lglyceraldehyde
(R,S) System
v Ultraviolet spectral properties of amino acids: Only phe, try, trp absorb UV light above 250 nm
v Separation of amino acids
Ion exchange
› Anion exchangers: Matrix is positively charged so anions bind
› Cation exchangers: Matrix is negatively charged so cations bind

Chapter 4 . Amino Acids
Chapter Objectives
It is imperative to learn the structures and one letter codes for the amino acids. Amino acids
are key molecules, both as components of proteins and as intermediates to important
biomolecules. The structures are shown in Figure 4.3 of Biochemistry. Figure 4.3 is presented
on the following two pages.
Classification of Amino Acids
Here is an alternative classification scheme.
Acidic amino acids and their amides:
aspartic acid, asparagine, glutamic acid, glutamine.
Basic amino acids:
histidine, lysine, arginine.
Aromatic amino acids:
phenylalanine, tyrosine, tryptophan.
Sulfur containing amino acids:
cysteine, methionine.
Imido acid:
proline.
Hydrophobic side chains:
glycine, alanine, valine, leucine, isoleucine.
Hydroxylic amino acids:
serine, threonine, (tyrosine).
To memorize the structures of the 20 amino acids, here are some aids:
The side chain of glycine is H, that of alanine is a methyl group, CH3.
The side chain of valine is just the methyl side chain of alanine with two methyl groups
substituting for two Hs. (In effect it is just dimethylalanine.)
The side chain of leucine is just valine with a -CH2- group between the valine side chain and the
α carbon (or, leucine is alanine with the valine side chain attached).
Isoleucine is an isomer of leucine with one of the methyl groups exchanged with a H on the
carbon attached to the α carbon.
Several of the amino acids have alanine as a base.
Phenylalanine has a phenyl group (i.e., benzene ring) attached to alanine.
Tyrosine is hydroxylated phenylalanine.
Serine has a hydroxyl group substituting for a H on the methyl group of alanine.
Cysteine can be thought of as the sulfur variety of serine i.e., with a sulfhydryl group in place of
the hydroxyl group of serine.
Threonine, a hydroxylic amino acid, can be thought of as methylated serine.
The acidic amino acids and their amides are quite easy to remember. Aspartic acid and
glutamic acid have carboxylic acid groups attached to -CH2- and (-CH2-)2, respectively. The
amides of these amino acids, namely asparagine and glutamine, have ammonia attached to the
side chain carboxyl groups in amide linkage.
The basic amino acids all have six carbons, counting the carboxyl carbon and the α−carbon.
Lysine's side chain is a linear chain of four -CH2- groups with a terminal amino group.
Arginine has a linear side chain of three -CH2- groups to which a guanidinium group is
attached. (The sixth carbon is the central C of the guanidinium group.) Guanidinium is like
urea but with a nitrogen group replacing the carbonyl oxygen. Histidine has an imidazole ring
attached to alanine. The ring contains two nitrogens and three carbons, a five-membered ring.
An easy way to remember the structure of proline is to know that it derives from glutamic acid.
The carboxyl side chain forms a C-N bond to the amino group to form this imido acid. (In the
process, the carbon is reduced so the side chain carboxyl oxygens do not show up in proline.)
44

Chapter 4 . Amino Acids
Methionine is a sulfur-containing amino acid. Biologically, it is also used as a methyl group
donor in the molecule S-adenosylmethionine (SAM). These two facts indicate that the side chain
ends in a S-CH3, which is attached to (-CH2-)2.
The remaining amino acid is tryptophan. Its side chain is an indole ring attached to a -CH2-
group. In effect it can be thought of as alanine with a indole ring. Substituted indole rings are
widely used in biochemistry. The basic structure is a six-membered benzene ring fused to a
five-membered ring containing a single N.
Nomenclature
The one letter codes for the amino acids must be memorized. In journals, the one letter
codes are used to show amino acid sequences. See the answer to Problem 2 for a discussion of
how the one letter codes are organized.
Chirality
The α−carbons of amino acids, excepting Gly, have four different groups attached to them,
and hence they are chiral carbons. Two different arrangements of the four groups about the
chiral carbon can be made, giving rise to two structural isomers. Structural isomers that are
mirror images of each other are called enantiomers. When two or more chiral centers exist on a
single molecule, 2 n structural isomers are possible, where n = the number of chiral centers. The
various structural isomers include enantiomers (mirror-image-related isomers) and non mirrorimage-related
isomers known as diastereomers.
The structural isomers can be distinguished from each other using the R and S prefixes
assigned to each chiral center of a molecule. The R and S prefixes are assigned according to a
set of sequence rules. The four groups about a chiral center are assigned priority numbers with
the highest priority given to the atom with the highest atomic number, thus S > O > N > C > H
(for elements found in amino acids). When two or more groups have the same primary element,
then elements attached to the primary element are considered. So for example, SH > OR > OH >
NH2 > COOH > CHO > CH2OH > CH3. Once priority assignments are made, the molecule is
viewed with the group having the lowest priority away from the reader. Moving from highest to
lowest priority, for the three remaining groups, clockwise movement indicates R and
counterclockwise indicates S configuration.
UV-absorbing Properties
Only three amino acids, phenylalanine, tyrosine, and tryptophan, absorb light in the near
UV range (i.e., 230 nm to 310 nm). These amino acids will dominate the UV absorption spectra
of proteins. The wavelength maxima for tyrosine and tryptophan are around 280 nm. In
addition, these two amino acids have extinction coefficients quite a bit larger than the extinction
coefficient of phenylalanine, whose λ max = 260 nm (See Figure 4.15). Thus very many proteins
have maximum absorbance around 280 nm. In contrast, nucleic acids absorb light with a
maximum around 260 nm due to the nucleotides. The extinction coefficients of the nucleotides
are larger than those for the aromatic amino acids. In general, nucleic acid contamination of a
protein sample will contribute to UV absorption at 260 nm. Often the 260/280 ratio is used as
a criterion for purity.
45

Chapter 4 . Amino Acids
Figure 4.3 The amino acids that are the building blocks of most proteins can be classified as (a)
nonpolar (hydrophobic), (b) polar, neutral, (c) acidic, or (d) basic. Also shown are the one-letter
and three-letter codes used to denote amino acids. For each amino acid, the ball-and-stick (left)
and space-filling (right) models show only the side chain.
46

Chapter 4 . Amino Acids
47

Chapter 4 . Amino Acids
48

Chapter 4 . Amino Acids
Problems and Solutions
1. Without consulting chapter figures, draw Fisher projection formulas for glycine,
aspartate, leucine, isoleucine, methionine, and threonine.
Answer:
Glycine
+ H3N
COO-
C
H
H
Aspartate
COO-
+ H3N C H
CH 2
COO-
Leucine
COO-
+
H3N C H
CH 2
CH
H3C CH3 49
Isoleucine
COO-
+
H3N C H
H 3C
C
H
CH 2
CH 3
Methionine
COO-
+
H3N C H
CH 2
CH 2
S
CH 3
Threonine
COO-
+
H3N C H
2. Without reference to the text, give the one-letter and three-letter abbreviations for
asparagine, arginine, cysteine, lysine, proline, tyrosine, and tryptophan.
Answer: For many of the amino acids the l letter code is the first letter of the amino acid.
Problems arise when two amino acids start with the same letter. Whenever this occurs, the
amino acid with the lowest molecular weight is assigned the letter. The other amino acid is
given a phonetic letter or the closest unused letter in the alphabet. The amino acids assigned
their first letters are: Glycine, Alanine, Valine, Leucine, Isoleucine, Serine, Threonine, Proline,
Cysteine, Methionine and Histidine. Aspartic acid is D, an easy way to remember it is to
pronounce it as "asparDic" acid. Glutamic acid is E, E follows D, glutamic acid follows aspartic
acid in chemical structure. E is also the closest unused letter to G. F is for phenylalanine,
(Fenylalanine). R is for arginine, remembered by pronouncing arginine (Rginine). N is for
asparagine; remember to stress the N sound. Glutamine follows asparagine, its l letter code is
the next unused consonant after N, namely Q. Lysine is K, the closest unused letter to L.
Tyrosine is Y; Y is the dominant sound in tyrosine. Tryptophan is W; remember "double ring,
double you".
3. Write equations for the ionic dissociations of alanine, glutamate, histidine, lysine,
and phenylalanine.
Answer: Alanine dissociation:
COOH
Glutamate Dissociation:
+
H3N C H
COOH
+ H3N C H
CH 2
CH 2
COOH
CH 3
COO-
+ H3N C H
CH 2
CH 2
COOH
COO-
+
H3N C H
CH 3
COO-
+ H3N C H
CH 2
CH 2
COO-
NH 2
COO-
C
H
CH 3
NH 2
H
COO-
C
H
CH 2
CH 2
C
COO-
CH 3
OH

Chapter 4 . Amino Acids
Histidine dissociation:
COOH
+ H3N C H
+HN
CH 2
NH
Lysine dissociation:
COOH
+ H3N C
CH 2
CH 2
CH 2
H
CH2 + H3N
Phenylalanine dissociation:
COOH
+H 3N C H
CH 2
+ H3N C H
+HN
+ H3N C
CH 2
CH 2
CH 2
CH 2
+ H3N
COO-
CH 2
NH
COO-
50
+ H3N C H
N
H H 2N C
COO-
+H 3N C H
CH 2
CH 2
CH 2
CH 2
CH 2
+ H3N
COO-
CH 2
NH
COO-
H
H 2N C H
N
H 2N C
COO-
H 2N C H
CH 2
COO-
CH 2
CH 2
CH 2
CH 2
CH 2
NH 2
NH
COO-
4. How is the pKa of the a-NH3 + group affected by the presence on an amino acid of the
a-COOH?
Answer: The pKa on an isolated amino group is around 10 (as for example, the side chain amino
group of lysine whose pKa = 10.5 (see Table 4.1). In general, the pKas of α amino groups in the
amino acids are around 9.5. Thus, the proximity of the α carboxyl group lowers the pKa of the
α amino group.
5. Draw an appropriate titration curve for aspartic acid, labeling the axis and
indicating the equivalence points and the pKa values.
Answer: For a titration curve the independent variable is the amount of acid or base used to
titrate the amino acid. The dependent variable is pH.
H

Chapter 4 . Amino Acids
The equivalence point is a point on the titration curve reached after a molar equivalent of acid or
base is added to the amino acid. For aspartic acid there are actually three equivalence points at
around 3.0, 6.2 and 12.2.
6. Calculate the concentrations of all ionic species in a 0.25 M solution of histidine at
pH 2, pH 6.4, and pH 9.3.
Answer: The pKas for histidine are 1.8, 6.0, and 9.2. At pH 2.0, we can ignore the two higher
pKas. At this pH, histidine's side chain and the α-amino group are protonated and thus both are
positively charged. So, the ionic species of histidine at pH = 2.0 depend critically on the
ionization state of the carboxyl group. When the carboxyl group is in the carboxylate form,
histidine will be in the 1+ ionic form. When the carboxyl group is protonated, and hence
uncharged, histidine will carry a 2+ charge. For the α-carboxyl group:
pH = pK a + log [COO − ]
[COOH] or,
[COO − ]
[COOH] = 10pH− pK a = 10 2.0−1.8 = 1.58 or [COO − ] =1.58 ×[COOH] and,
[COO − ] +[COOH] = 0.25M
Thus, 1.58 ×[COOH] +[COOH] = 2.58[COOH] = 0.25M and,
[COOH] = .25M
2.58
= 0.097M and,
[COO − ] = 1.58 ×[COOH] = 0.153M
So, at pH = 2.0, [His 2+ ] = 0.097M and [His 1+ ] = 0.153M
The concentration of uncharged histidine and negatively charged histidine are both small but
can be estimated as follows. The concentration of uncharged histidine will be approximately
equal to the concentration of the unprotonated side-chain species. Using the Henderson-
Hasselbalch equation with pKa = 6.0, pH = 2.0, and [His 1+] = 0.153 M, we find that
51

Chapter 4 . Amino Acids
pH = 6.0 + log [His 0 ]
[His 1+ ]
52
or,
[His 0 ]
[His 1+ = 10
]
pH − 6.0 = 10 2.0− 6.0 and,
[His 0 ] = [His 1+ ] × 10 −4 = 1.53 × 10 −5 M
The concentration of negatively charged histidine is calculated using the Henderson-Hasselbalch
equation with pKa = 9.2, pH = 2.0, and [His 0] = 1.53 x 10 -5 M. In this case
pH = 9.2 + log [His1- ]
[His 1- ]
[His 0 ]
[His 0 ]
or,
= 10 pH− 9.2 = 10 2.0−9.2 and,
[His 1- ] = [His 0 ] ×10 −7.2 = 1.53 ×10 −5 ×10 −7.2 = 9.6 ×10 −13 M
At pH = 2.0 we have: [His 2+] = 0.097 M , [His 0] = 1.53 x 10 -5 M,
[His 1+] = 0.153 M, [His 1-] = 9.6 x 10 -13 M.
At pH = 6.4, we can assume that the carboxyl group is fully unprotonated and carries a -1
charge while the α-amino group is fully protonated and carries a +1 charge. Thus, the ionic
species of histidine depends critically on the ionic state of the imidazole side chain. When the
imidazole group is protonated, histidine carries a net +1 charge. When it is unprotonated,
histidine is uncharged.
So, using
pH = 6.0 + log [His 0 ]
[His 1+ ]
and [His 0 ] + [His 1+ ] = 0.25M ,
we find that the ionic species of histidine at pH = 6.4 are :
[His 0 ] = 0.179M and [His 1+ ] = 0.071M
[His 2+] and [His 1-] are calculated using the Henderson-Hasselbalch equation with pH = 6.4, pKa
= 1.8 and 9.2 and [His 1+] = 0.071 M and [His 0] = 0.179 M. We find [His 2+] = 2.8 x 10 -6 M and
[His 1-] = 2.25 x 10 -4 M.
At pH = 6.4 we have: [His 2+] = 1.78 x 10 -6 M , [His 0] = 0.179 M,
[His 1+] = 0.071 M, [His 1-] = 2.84 x 10 -4 M.
At pH = 9.3, the carboxyl group is unprotonated and negatively charged, the imidazole group is
unprotonated and uncharged, and only the protonation state of the α-amino group needs to be
determined. Histidine, at pH = 9.3 will be portioned between the His 0 and His 1- states. From
pH = 9.2 + log [His1- ]
[His 0 ]
and [His 0 ] + [His 1- ] = 0.25M ,
we find that the ionic species of histidine at pH = 9.3 are :
[His 0 ] = 0.111M, [His 1- ] = 0.139M and
[His 2+ ] = 1.75 ×10 -12 M, [His + ] = 5.5 ×10 -5 M
7. Calculate the pH at which the g carboxyl group of glutamic acid is two-thirds
dissociated.
Answer: The pKa of γ carboxyl group of glutamic acid is 4.3.

Chapter 4 . Amino Acids
Using the Henderson - Hasselbalch equation
pH = pK COOH + log [C γ OO− ]
[C γ OOH]
When the γ - carboxyl group is two - thirds dissociated :
[C γOO − ]
[CγOO − =
] +[C γOOH]
2
3 or, [C γOO − ] = 2
3 [C γOO − ⎛ ] +[C ⎞
⎝
γOOH]
⎠
Thus, [C γOOH] = 1
2 [C γOO − ]
Upon substituting into the Henderson-Hasselbalch equation we find :
pH = 4.3 + log [C γOO − ]
[Cγ OOH] = 4.3 + log [C γOO − ]
1
2 [C γOO − ]
pH = 4.6
53
= 4.3 + log 2
8. Calculate the pH at which the e-amino group of lysine is 20% dissociated.
Answer: For the lysine side chain, pKa = 10.5.
Using the Henderson - Hasselbalch equation
pH = pK +
NεH + log
3
[N εH2] +
[N εH3 ]
When the ε - amino group is 20% d issociated:
[N εH2] = .2 (20%) or, [N
+ εH2] = .2 ⎛
+
[N εH2] +[N εH3 ] ⎞
⎝
⎠
[N εH2 ] +[N εH3] Thus, [N εH2] = 1
4 [N +
εH3 ]
Upon substituting into the Henderson-Hasselbalch equation we find :
pH = 10.5 + log [N 1
εH2 ]
=10.5 + log
4
+
[N εH3 ]
[N +
εH3 ]
+
[N εH3 ]
pH = 9.9
= 10.5 − log 4
9. Calculate the pH of a 0.3 M solution of (a) leucine hydrochloride, (b) sodium leucinate,
and (c) isoelectric leucine.
Answer: The solution 0.3 M leucine HCl is composed of 0.3 M leucine and 0.3 M HCl. Thus, we
expect it to have an acidic pH and only the α carboxyl group of leucine is involved in proton
equilibrium.
The pKa of the α carboxyl of leucine is 2.4. Thus,
pH = pKa + log [ A − ]
[HA] = 2.4 + log [COO− ]
[COOH]
[COO − ] + [COOH ] = 0.3M (2)
For charge neutrality :
[Cl - ] + [COO - ] = [H + ] + [NH 3 + ]
(1) and,

Chapter 4 . Amino Acids
If we recognize that [Cl - ] =[NH 3 + ] (because Cl - and the amino acid are in equimolar amounts),
the charge neutrality equation may be simplified to:
[COO - ] =[H + ] and combined with (1) and (2) we have :
[H
pH = 2.4 + log
+ ]
0.3−[H + ]
If we make the assum ption that [H + ]

Chapter 4 . Amino Acids
At the pI, [ COO − ] = [NH 3 + ]
Using the Henderson - Hasselbalch equations
pH = pKCOOH + log [COO− ]
[COOH] and pH = pK + + log
NH 3
[NH2] +
[NH 3 ]
and recognizing that [COOH] = [Amino acid] total -[COO − ]
and that [NH 2] = [Amino acid] total -[NH 3 + ]
upon substituting, solving for [ COO − +
] and [NH 3],
and setting these terms
equal at pH = pI we find that
pI = pKCOOH + pK +
NH 3
2
2.4 + 9.6
pI = = 6.0
2
or
10. Quantitative measurements of optical activity are usually expressed in terms of the
specific rotation, [a]D 25, defined as
[ α ] D 25 = Measured rotation in degrees × 100
(Optical path in dm) ×(conc. in g/ml)
For any measurement of optical rotation, the wavelength of the light used and the
temperature must both be specified. In this case, D refers to the "D line" of sodium at
589 nm and 25 refers to a measurement temperature of 25°C. Calculate the
concentration of a solution of L-arginine that rotates the incident light by 0.35° in an
optical path length of 1 dm (decimeter).
Answer:
[α ] D 25 = Measured rotation in degrees × 100
(Optical path in dm) ×(conc. in g/ml)
[Arg] = 0.35°×100
[α ] D 25 × 1dm
[α ] D 25 for L-arginine = 12.5° (from Table 4.2)
Thus, [Arg] = 0.35°×100
= 2.8 g/ml
12.5°× 1dm
11. Absolute configurations of the amino acids are referenced to D- and Lglyceraldehyde
on the basis of chemical transformations that can convert the molecule
of interest to either of these reference isomeric structures. In such reactions, the
stereochemical consequences for the asymmetric centers must be understood for each
reaction step. Propose a sequence of reactions that would demonstrate that L(-)-serine is
stereochemically related to L(-)-glyceraldehyde.
Answer: The aldehyde group of L(-)-glyceraldehyde is converted to L-glyceric acid by oxidation
and L-glyceric acid is converted to 2-hydroxypropanoic acid. 2-Hydroxypropanoic acid is
converted to 2-bromopropanoic acid by nucleophilic substitution using concentrated HBr. The
reaction results in alteration in stereochemistry. 2-Bromopropanoic acid is subsequently
converted to L-(-)-alanine by ammonolysis and alteration in stereochemistry. Finally, L-(-)alanine
is convert serine. The reaction sequence is shown below.
55

Chapter 4 . Amino Acids
L-(-)-Glyceraldehyde L-Glyceric acid 2-Hydroxypropanoic acid
CHO
HO C H
CH2OH
COOH
H C Br
CH3
COOH
HO C H
CH2OH
COOH
H2N C H
CH3
56
COOH
HO C H
CH3
COOH
H2N C H
CH2OH
2-Bromopropanoic acid L-(-)-Alanine L-(-)-Serine
12. Describe the stereochemical aspects of the structure of cystine, the structure that is
a disulfide-linked pair of cysteines.
Answer: Cystine (dicysteine) has two chiral carbons, the two a-carbons of the cysteine moieties.
Since each chiral center can exist in two forms there are 4 stereoisomers of cystine. However, it
is not possible to distinguish the difference between L-cysteine/D-cysteine and D-cysteine/Lcysteine
dimers. So three distinct isomers are formed.
H
H
H
H
-OOC
+H 3N
C
CH 2
S
S
CH2 C COO-
H
NH 3+
L-cysteine-
L-cysteine
+H 3N
+H 3N
C
CH 2
S
S
CH2 C COO-
H
COO-
L-cysteine-
D-cysteine
-OOC
-OOC
C
CH 2
S
S
CH2 C NH3+ H
NH 3+
D-cysteine-
L-cysteine
+H 3N
-OOC
C
CH 2
S
S
CH 2
C NH3+ H
COO-
D-cysteine-
D-cysteine
13. Draw a simple mechanism for the reaction of a cysteine sulfhydryl group with
iodoacetamide.
Answer:
+H 3N
I CH2 C NH2 S-
CH2 C COO-
H
O
I -
+H 3N
NH2 C O
CH 2
S
CH2 C COO-
14. Describe the expected elution pattern for a mixture of aspartate, histidine,
isoleucine, valine, and arginine on a column of Dowex-50.
H

Chapter 4 . Amino Acids
Answer: Dowex is an anion exchanger with a positive charge due to a quaternary amine. Thus,
one expects amino acids to elute from Dowex in the following order: basic (positively-charged)
amino acids; neutral (uncharged amino acids); acidic (negatively-charged amino acids).
We expect arginine to elute first followed by histidine. Of these two basic amino acids, arginine
will have a +1 charge at neutral pH whereas histidine’s charge will slightly less that +1.
Next we expect isoleucine followed by valine. The charge on these amino acids will be close to
(but not) zero; however, isoleucine will be slightly less negatively charged than valine. (The
isoelectric point, the pH at which a molecule has a net charge of zero, is simply the average
value of the pKas (for amino acids lacking ionizable R groups). Thus, isoleucine’s isoelectric
point is (2.4 + 9.7)/2 = 6.05 whereas valine’s isoelectric point is (2.3 + 9.6)/2 = 5.95. At neutral
pH we expect isoleucine to be slightly less negatively charged than valine.)
Finally, we expect aspartate to elute last because it is an acidic, negatively-charged amino acid.
15. Assign (R,S) nomenclature to the threonine isomers of Figure 4.14.
Answer:
+H 3N
H
57
COO-
The groups about the α-carbon of L-threonine are assigned the following priority: NH3 + > COO- >
Cβ> H. In Fischer projections, two groups joined to the α-carbon (i.e., NH3 + and H) are coming
out of the plane while the other two groups are into the plane. By rotating the molecule such
that the group with the lowest priority, namely H, is away from the reader, the NH3 + group will
be located on the right side of the α-carbon, the carboxyl group above and to the left, and the
remaining β-carbon to the left and below the α-carbon:
3
C
C
H
CH 3
2
OH
COO - H
C C C α NH +
β 3
Since moving from highest to lowest priority involves counterclockwise movement the α-carbon
is in the S configuration.
For the β-carbon, the priority is OH > Cα > CH3 > H. The orientation of the Cα-Cβ bond was
already defined for the α-carbon as being into the plane therefore it must be out of the plane for
the β-carbon, as must be the bond to -CH3. Thus, bonds to OH and H are into the plane. By
rotating the molecule such that H is behind the β-carbon, we find the OH to the left, CH3 below
and Cα above:
HO
1
Cα
C
β
Since moving from highest to lowest priority involves clockwise movement the α-carbon is in the
R configuration. The β-carbon is in the R configuration.
2
1
H
CH3
3

Chapter 4 . Amino Acids
+H 3N
H
COO-
C
C
H
CH 3
OH
H
HO
COO-
C
C
CH 3
NH 3+
H
58
+H 3N
COO-
C
C
CH 3
H
H
H
COO-
C
C
CH 3
NH 3+
L-Threonine D-Threonine L-Allothreonine D-Allothreonine
By inspection we see that
L-threonine is (2S, 3R) threonine
D-threonine is (2R,3S) threonine
L-allothreonine is (2S,3S) threonine
D-allothreonine is (2R,3R) threonine
Questions for Self Study
1. Fill in the blanks: A bond is an amide bond between two . The bond is formed by a
reaction between the of one amino acid and the of a second amino acid with the
elimination of the elements of . The result is a linear chain with an end and a end.
The chain can be extended into a polymer with additional amino acids. The polymer formed is
called either a or a depending on the number of amino acids joined. The amide bond
can be broken by the addition of the elements of water across it in a reaction.
2. List the four classes of amino acids.
3. Indicate which class of amino acids best represents the statements presented below:
a. Their side chain may contain a hydroxyl group. .
b. Negatively charged at basic pH. .
c. Relatively poorly soluble in aqueous solution. .
d. Relatively soluble in aqueous solution. .
e. Positively charged at acidic pH. .
f. Side chains are capable of hydrogen bonding. .
g. Includes the amino acids arginine, histidine and lysine. .
h. Side chains contain predominantly hydrocarbons._____
i. Side chains are responsible for the hydrophobic cores of globular proteins. .
j. The amides of glutamic and aspartic acid belong to this group. .
4. From the list of amino acids presented indicate which one best fits the description:
a. The smallest amino acid (It also lacks a stereoisomer).
alanine, glycine, phenylalanine, glutamine
b. An aromatic amino acid.
glutamic acid, tyrosine, isoleucine, proline
c. A sulfur-containing amino acid.
methionine, aspartic acid, arginine, leucine
d. An amino acid capable of forming sulfur-sulfur bonds.
methionine, proline, cysteine, tryptophan
e. A branched chain amino acid.
methionine, leucine, aspartic acid, asparagine
5. Fill in the blanks. Most of the amino acids are chiral compounds because they have at least
one carbon atom with different groups attached. Thus, there are ways of arranging the
four groups about the carbon atom. For the case of an amino acid with a single asymmetric
carbon, the isomers pairs that can be formed are called ; they are nonsuperimposable, .
images of each other. Using the D,L system of nomenclature, the commonly occurring amino
acids are all amino acids.
6. Which three amino acids absorb light in the ultraviolet region of the spectrum above 250
nm?
HO
H
OH

Chapter 4 . Amino Acids
7. Of the following techniques which are commonly used to separate amino acids: ion
exchange, NMR, electroporation, HPLC, UV spectroscopy, ninhydrin reaction?
8. DTNB (Ellman's reagent), iodoacetate, and N-ethylmaleimide are three agents that are used to
modify which amino acid? What is the reactive group on the amino acid with which all three
agents react?
9. Although there are twenty common amino acids, there are hundreds of different amino acids
found in nature, some of which derive from the common amino acids by simple chemical
(biochemical) modification. Name one common modification of amino acids found in nature.
10a. Of the twenty common amino acids which amino acid has a pKa around neutrality?
b. What amino acid lacks an amino group?
Answers
1. peptide; amino acids; amino group; carboxyl group; water; N-terminal (or amino ); Cterminal
(or carboxyl); polypeptide; protein; hydrolysis.
2. Nonpolar or hydrophobic; neutral polar; acidic; basic.
3. a./neutral polar; b./acidic; c./nonpolar; d./neutral polar, acidic, and basic; e./basic;
f./neutral polar, acidic, and basic; g./basic; h./nonpolar; i./nonpolar; j./neutral polar.
4. a. glycine; b. tyrosine; c. methionine; d. cysteine; e. leucine.
5. Four; two; enantiomers (or mirror image isomers); mirror; L.
6. Phenylalanine, tyrosine, and tryptophan.
7. ion exchange, HPLC
8. cysteine; sulfhydryl group.
9. Hydroxylation, methylation, carboxylation, phosphorylation.
10. a./Histidine; b./proline.
Additional Problems
1. Sketch the titration curve of histidine.
2. In the protein hemoglobin, a number of salt bridges (ionic bonds) are broken in going from
oxygenated to deoxygenated hemoglobin. One in particular involves an aspartic acid: histidine
ionic interaction. (a). At pH = 7.0, would you expect this salt bridge to form? Explain. (b).
Under certain conditions (low oxygen tension) the salt bridge does form. Explain how the
influence of aspartic acid on histidine is similar to the influence of the α carboxyl group on the
pKa of the α amino group (and vice versa).
3. How might the presence of calcium ions affect the apparent pKas of the side chains of aspartic
acid and glutamic acid?
4a. The ultraviolet spectra of proteins are largely determined by what three amino acids?
b. In working with nucleic acids it is often useful to measure the optical densities at 260 nm
and 280 nm. Nucleic acids (DNA and RNA) absorb more strongly at 260 nm than 280 nm such
that the ratio A260 : A280 ranges from 1.6 to 2.0. What might happen to the A260 : A280 ratio of a
solution of nucleic acid contaminated with protein?
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Chapter 4 . Amino Acids
c. Proteins typically have a A260 : A280 ratio less than one. Using Figure 4.15, estimate the A260 :
A280 ratio of a protein whose amino acid composition includes two moles of Trp and one mole of
Tyr per mole of protein.
d. Certain proteins lack both Trp and Tyr yet still absorb ultraviolet light in the range of 240 nm
to 270 nm. What amino acid is responsible for this? How would the A260 : A280 ratio of a
protein rich in this amino acid differ from a typical protein containing Trp and Tyr?
5. Poetry comes in many forms. Can you write a short, two line poem, using the single-letter
abbreviations for the amino acids?
Abbreviated Answers
1. Histidine is the only amino acid with a pKa around neutrality. The pKas of histidine are 1.8,
6.0, and 9.2. The titration curve will be similar to the one shown in the answer to problem 5
but with an additional plateau around pH = 6.0.
2. (a) The pKas of the side chains of aspartic acid and histidine are 3.9 and 6.0 respectively. At
pH = 7.0, the side chain of aspartic acid is unprotonated and therefore negatively charged,
whereas the side chain of histidine is also unprotonated and therefore uncharged. We might not
expect this interaction to occur.
(b) The fact that the salt bridge forms indicates that the two amino acid side chains are close
together in the three dimensional structure of the protein. The proximity of the negatively
charged aspartic acid must therefore raise the pKa of the histidine side group.
3. Calcium (Ca 2+) may bind to carboxylate ions. Therefore, the presence of calcium may lower
the apparent pKas of aspartic acid and glutamic acid.
4. a./Phe, Tyr, and Trp.; b./ A260 : A280 ratio may be lower.
c./The molar absorptivity for Trp at 280 nm and 260 nm are approximately 4,000 and 3,000
respectively. The molar absorptivity for Tyr at 280 nm and 260 nm are approximately 1,000 and
500 respectively. The A260 : A280 ratio is given by:
(2 × 3,000) + 500
Ratio = = 0.72
(2 × 4,000) +1, 000
d./Phe. The A260 : A280 ratio would be much greater that 1.
5. Here is a start that is by no means original. "I think that I shall never see a ...." Try
composing one with the nucleoside one-letter code (A,T,G and C) if you really want a challenge.
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Chapter 4 . Amino Acids
Summary
Amino acids are the building blocks of proteins. There are 20 amino acids commonly found
in proteins. The general structure of an amino acid is based on a central α-carbon, to which is
attached a carboxyl group, a hydrogen, an amino group, and a variable side chain or R group.
Amino acids can be classified according to the nature of their R groups into three classes. The
nonpolar, hydrophobic amino acids include alanine, valine, leucine and isoleucine in addition
to proline, methionine, phenylalanine and tryptophan. Polar, uncharged amino acids
include glycine, serine, threonine, tyrosine, asparagine, glutamine, and cysteine. The
acidic amino acids are aspartic acid and glutamic acid and the basic amino acids include
histidine, lysine, and arginine. The amino acids are weak polyprotic acids. The carboxyl
group is a rather strong carboxylic acid with pKa around 2.0, whereas the amino group is a base
with pKa around 9.5. At neutral pH, the carboxyl group is unprotonated and negatively charged
whereas the amino group is protonated and positively charged. Considering only these two
groups at pH = 7.0, amino acids are zwitterionic. Several of the side chains have titratable
groups including aspartic acid, glutamic acid, cysteine, tyrosine, arginine, histidine, and lysine.
In proteins, amino acids are joined by peptide (or amide) bonds. The carboxyl group of one
amino acid is joined to the amino group of another amino acid, with elimination of the elements
of water.
In addition to the twenty common amino acids, there are a large number of modified amino
acids found in nature. Examples include hydroxylation of lysine and proline, methylation of
lysine, carboxylation of glutamic acid, phosphorylation of serine, threonine and tyrosine.
Metabolites derived from amino acids play key roles in cells. Knowing the structures of
ornithine, citrulline, aspartic acid and arginine will serve as a strong basis for understanding the
urea cycle. Transamination of glutamic acid and aspartic acid produce α-ketoglutarate and
oxaloacetate, respectively, two citric acid cycle intermediates. The same reaction with alanine
produces pyruvate. A great many small molecules acting as neurotransmitters, hormones,
antibiotics, and other roles are directly derived from amino acids.
The amino acids can participate in a number of useful chemical reactions, some specific to
either the carboxyl group or the amino group common to all amino acids. Other chemistries are
specific for side groups. For example, the sulfhydryl group of cysteine can be modified with Nethylmaleimide
or iodoacetate. It will react with DTNB in a reaction producing a colored
compound, which can be readily quantitated. The amino group of lysine, hydroxyl groups of
serine, tyrosine and threonine and the carboxyl groups of aspartic and glutamic acid can
participate in specific reactions.
The α-carbons of all of the amino acids except glycine are chiral. Thus amino acids can exist
as stereoisomers. In nature, the L amino acids predominate. We will consider stereochemistry
again when sugar structures are discussed.
There are two general methods for separating and analyzing amino acid mixtures. In one
method, the ionic properties of the amino acids are exploited in ion exchange. Ion exchange
involves a stationary charged species in equilibrium with a counter ion. Conditions are
established such that the amino acids can displace the counter ion and bind to the stationary
phase. The degree to which amino acids bind to the stationary phase depends on their intrinsic
charge and on the concentration of counter ion with which they compete. Removal of amino
acids from the stationary phase can be accomplished either by changing the pH of the solution
(and thus changing the charge on the amino acids) or increasing the concentration of counter
ion. The conditions under which a particular amino acid is displaced from the stationary phase
are characteristic of the amino acid.
The other technique for separating amino acids is to react the amino terminus with a
hydrophobic group and to separate the derivatized amino acids by reverse phase
chromatography. In reverse phase chromatography, derivatized amino acids are bound to a
stationary, hydrophobic phase and subsequently eluted with increasing concentrations of a
polar substance (e.g., methanol).
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